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4 Recent Patents on Anti-Cancer Drug Discovery, 2012, 7, 4-13 Expanding Targets for a Metabolic Therapy of Cancer: L-Asparaginase Daniele Covinia, Saverio Tarditob, Ovidio Bussolatib, Laurent R. Chiarellic, Maria V. Pasquettoa, Rita Digiliod, Giovanna Valentinic and Claudia Scotti*,a a Department of Experimental Medicine, Section of General Pathology, University of Pavia, Via Ferrata 1, 27100 Pavia, Italy; bDepartment of Experimental Medicine, Unit of General and Clinical Pathology, University of Parma, Via Volturno 39, 43125 Parma, Italy; cDepartment of Biochemistry, “A. Castellani”, University of Pavia, Via Taramelli 3/b, 27100 Pavia, Italy; d Centre for Innovation and Technology Transfer, University of Pavia, Corso Strada Nuova 65, 27100 Pavia, Italy Received: March 31, 2011; Accepted: April 15, 2011; Revised: April 28, 2011 Abstract: The antitumour enzyme L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1, ASNase), which catalyses the deamidation of L-asparagine (Asn) to L-aspartic acid and ammonia, has been used for many years in the treatment of acute lymphoblastic leukaemia. Also NK tumours, subtypes of myeloid leukaemias and T-cell lymphomas respond to ASNase, and ovarian carcinomas and other solid tumours have been proposed as additional targets for ASNase, with a potential role for its glutaminase activity. The increasing attention devoted to the antitumour activity of ASNase prompted us to analyse recent patents specifically concerning this enzyme. Here, we first give an overview of metabolic pathways affected by Asn and Gln depletion and, hence, potential targets of ASNase. We then discuss recent published patents concerning ASNases. In particular, we pay attention to novel ASNases, such as the recently characterised ASNase produced by Helicobacter pylori, and those presenting amino acid substitutions aimed at improving enzymatic activity of the classical Escherichia coli enzyme. We detail modifications, such as natural glycosylation or synthetic conjugation with other molecules, for therapeutic purposes. Finally, we analyse patents concerning biotechnological protocols and strategies applied to production of ASNase as well as to its administration and delivery in organisms. Keywords: Acute lymphoblastic leukaemia, amino acid metabolism, asparagine, cancer, glutaminase, glutamine, L-asparaginase. INTRODUCTION Initial studies concerning L-asparaginase (ASNase) date back to the 1900s, when the enzyme was isolated from different animal sources [1] and the key biological role of Lasparagine (Asn) in metabolism was established. Only in the 1950s, the search for immune components able to prevent and/or halt tumour development lead to the discovery of the antibody- and complement-independent chemotherapeutic effect of guinea pig serum against murine Gardner lymphosarcoma [2, 3] and ASNase was then identified as the agent responsible for this effect [4-6]. The enzyme, isolated from many bacterial organisms, including Escherichia coli (E. coli) [7] and Erwinia carotovora (E. carotovora) [8], was then thoroughly characterised and produced in convenient recombinant systems, and it is nowadays a part of several standard chemotherapeutic protocols thanks to its confirmed anticancer activity. In nature, Asn is synthesised by asparagine synthetase (ASNsynt, EC 6.3.5.4) from aspartic acid and L-glutamine (Gln), and it is the substrate of ASNase (EC 3.5.1.1), which catalyses its deamidation giving L-aspartic acid and ammonia as reaction products. ASNase may also act on Gln thereby obtaining L-glutamic acid and ammonia. Bacterial ASNases are the best characterised members of this enzyme *Address correspondence to this author at the Department of Experimental Medicine, Section of General Pathology, University of Pavia, Via Ferrata, 1, 27100 Pavia, Italy; Tel: +39 0382 986335; Fax: +39 0382 986893; E-mail [email protected] 2212-3970/12 $100.00+.00 family. Type I ASNases are cytoplasmic, display high Km values vs. Asn and are also active towards Gln. Type II ASNases are periplasmic, exhibit low Km values vs. Asn, and have low-to-negligible activity towards Gln. These enzymes exhibit hyperbolic response to Asn [9, 10], though ASNase I from E. coli displays positive cooperativity towards Asn and is allosterically regulated by the substrate itself [11]. A minority of ASNases, also referred to as glutaminasesasparaginases (EC 3.5.1.38), transform either Asn or Gln into their corresponding acids with an activity against Gln 10 times lower than that exhibited against Asn [12]. Bacterial ASNases are 140-150kDa tetramers, more accurately described as dimers of intimate dimers [13], built up by identical subunits of 300-350 amino acid residues. Four independent catalytic sites are located at the intersubunit interface of the intimate dimers [13, 14]. The enzymatic activity is likely to depend on the classic two-step ping-pong mechanism of serine proteases and on two catalytic triads consisting, in E. coli ASNase, of Thr12-Tyr25-Glu283 and Thr89-Asp90-Lys162, respectively [10, 15]. Most of the information about this enzyme class derives from studies on E. coli ASNase: it works in a pH range between 5 and 10 [16], it has optimal ASNase activity between pH 8.0 and 9.0 and optimal glutaminase activity between pH 5.5 and 7.5 [17]. Its affinity constants (Km) for Asn and Gln are, respectively, 1.15x10-5 e 6.25x10-3 M; its isoelectric point is 5.2. The enzyme is not linked to either carbohydrates or phospholipids [18]. © 2012 Bentham Science Publishers Therapeutic L-Asparaginases Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 1 METABOLIC PATHWAYS TRIGGERED BY ASPARAGINASES Since ASNases catalyse the hydrolysis of Asn and Gln, the nutritional stress caused by depletion of these amino acids is reflected by the metabolic responses of treated cells. Both amino acids work as vehicles in nitrogen transport but their relative importance is widely different in different organisms, with Gln appearing much more important than Asn in mammals. Gln is the most abundant amino acid in human plasma, with concentrations ranging from 0.6 to 0.9mM, while Asn concentration is ten-fifteen times lower [19]. Moreover, although high variability is presumed among the various tissues, Gln is also the most abundant amino acid in the intracellular compartment, with tissue concentrations as high as 20mM [20]. While several widely used cell culture media are nominally Asn-free, it has been known since the early years of cell cultures that Gln is not dispensable for in vitro growth of mammalian cells [21]. However, the basis for this requirement has been never completely elucidated [22-24], since Gln is not considered an essential amino acid and it can be produced by most cultured cells. Thus, it not surprising that, so far, Gln depletion has been studied much more than Asn depletion in a variety of tissues and conditions. However, Asn depletion is per se able to induce ASNS, the gene for ASNsynt, and to prolong the shelf life of its mRNA [25], although these effects are not exclusive of this condition but are observed also upon the depletion of Gln as well as of any single essential amino acid [25] Fig. (1). Thus, although Asn and Gln are not essential, their depletion seems able to induce at least part of the complex transcriptional response elicited by complete amino acid deprivation [26]. Under such conditions, cells activate a kinase named General Control Nonderepressible 2 (GCN2), which phosphorylates the -subunit of the eukaryotic Initiation Factor 2 (eIF2, Fig. (1)). This phosphorylation represents a fundamental metabolic switch that lowers the rate of global protein synthesis, dependent on formation of the classical ternary activation complex, and leads to energy conservation needed for cell survival. At the same time, translation of a sub-population of mRNAs, endowed with Internal Ribosome Entry sites (IRES), is favoured. These mRNAs encode cell-defence proteins or proteins involved in apoptosis. For example, one of these proteins is transcription factor ATF4 Fig. (1), which, once translated, leads to expression of several genes involved in cell survival or, depending on environmental conditions, apoptosis, such as GADD153/CHOP. The direct role of GCN2 kinase in the response to ASNase has been recently demonstrated in vivo by Anthony’s group [27, 28]. Previous contributions of the same group had already demonstrated that treatment of mice with ASNase leads to enhanced eIF2 phosphorylation in liver, but not in pancreas, suggesting a tissue-specificity of the metabolic effects of the enzyme [29]. In the same report, using ASNases endowed with different glutaminase activities, it was also demonstrated that eIF2 phosphorylation was dependent on Gln, rather than Asn depletion, although both conditions are able to induce ASNS and CHOP [29]. Many of the responses elicited by ASNase treatment are directly referable to attempts of adaptation to Asn and/or Gln 5 L-Asparaginase Gln depletion Asn depletion GCN2 eIF2 GLNsynt p-mTOR mTOR p-eIF2 ATF4 ASNsynt SNAT2 ASCT1 Others Protein synthesis Cell Growth CHOP Others Apoptosis Fig. (1). A simplified scheme of the metabolic pathways triggered by ASNase. See text for details. shortage. For instance, in MOLT-4 leukaemia cells, E. coli ASNase promotes not only induction of ASNS, but also synthesis of ASCT1 and SNAT2, two sodium-dependent transporters for Asn and Gln [30], Fig. (1). Moreover, also the activity of Glutamine Synthetase (GLNsynt) is increased upon ASNase treatment, although, in this case, the site of regulation is post-transcriptional [30]. The role of GLNsynt in the metabolic adaptation to ASNase has been directly addressed in sarcoma cells, demonstrating that increase in GLNsynt activity parallels enhanced abundance of the enzyme, likely stabilised by intracellular depletion of Gln [31]. This adaptation is important for cell survival, since a GLNsynt inhibitor powerfully synergises the cytotoxic effects of ASNase [32, 33]. The relationships between effects of ASNase treatment and pathways triggered by amino acid starvation have also been strengthened by a genome-wide study of the response to ASNase (likely derived from E. coli, although this detail was not given) of human ALL cells [34]. In that study, Fine et al. found that more than 800 genes have their expression modified after exposure to the antitumour enzyme [34]. Among these, besides ASNS, genes for tRNA synthetases, amino acid transporters, ATF and CCAAT/enhancer-binding protein (C/EBP) families of transcription factors were found induced, while expression of genes associated with proliferation was suppressed [34]. Interestingly, ATF4 and members of the C/EBP family synergistically interact to promote induction of amino acid-responsive genes by binding to composite C/EBP-ATF response elements (CARE) [35]. Protein synthesis slowdown in ASNase-treated cells is not due only to enhanced eIF2 phosphorylation. Another important metabolic regulatory switch affected by the antitumour enzyme is the mTOR pathway, which controls cell growth and division, coupling permissive environmental conditions and protein synthesis rate. This pathway is activated on the basis of an integration of signals deriving from growth factors, energy status, nutrient availability, and the presence of stress of various nature (see [36] for a recent 6 Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 1 review). In the same report cited above [29], Reinert et al. demonstrate that mTOR signalling is repressed in liver and pancreas of ASNase-treated animal. Importantly, this effect was observed upon treatment with E. coli ASNase (endowed with glutaminolytic activity) but not with the enzyme from Wolinella succinogenes, which is nearly devoid of glutaminase activity. This result suggests that mTOR inhibition is a specific effect of ASNase-induced Gln depletion. Consistently, it is well known that mTOR activity is influenced by the availability of selected amino acids and that, in particular, Gln is needed for mTOR-dependent signalling [37]. The importance of Gln in tumour metabolism has boosted the interest towards the search for inhibitors of GLNsynt and glutaminase, and has renewed the attention to the therapeutic potential of glutaminases. MECHANISMS OF ANTITUMOUR EFFECTS The anti-tumour action of bacterial ASNases is primarily attributed to their ability to reduce Asn blood concentration causing a selective inhibition of growth of sensitive malignant cells [38, 39], Fig. (1). Despite the long standing experience with these drugs, however, the precise metabolic and molecular bases of ASNase antitumoural effect have not yet been fully elucidated. For example, cell sensitivity has been related to low or absent expression of ASNsynt [40, 41], and, in vitro, resistance to ASNase has been associated with upregulation of ASNsynt mRNA expression. Nevertheless, ASNsynt up-regulation does not correlate with early poor response to ASNase in children with ALL [42, 43]. Moreover, TEL/AML1 positive leukaemic cells are unable to progress into the S phase of cell cycle under nutrition stress caused by ASNase, despite their ability to upregulate ASNsynt [44]. The contribution of the glutaminolytic activity of ASNases to the biological effects of the enzyme is also a matter of discussion. The prevailing opinion is that Gln hydrolysis contributes to the toxic effects of ASNases [29, 45, 46]. However, Gln hydrolysis is also needed for an effective and sustained Asn depletion [47] and may contribute significantly to the antineoplastic activities of the enzyme [48]. CLINICAL APPLICATIONS Today, ASNase finds its main use in medicine, with some of the available commercial names being Elspar® (E. coli ASNase), Erwinase® (Erwinia chrysanthemi ASNase), and Oncaspar® (pegylated E. coli ASNase). At the moment of writing, the website of the National Institute for Health [49] lists 132 open clinical trials involving ASNase. Since the discovery of ASNase anticancer effect, bacteria have been the best source of enzyme. Among all bacteria analysed in the early years, E. coli and E. carotovora have shown the greater production of enzymes with good antitumour activity. Unluckily, the enzymes have also shown their toxicity, so that research regarding ASNase is still of great interest both for the clinics and for biotechnology. For over thirty years now, the enzyme has been part of established anticancer protocols for the treatment of acute lymphoblastic leukaemia (ALL) [50], but it has been used Covini et al. also for other types of haematological malignancies like myeloblastic leukaemia [51], Hodgkin and non-Hodgkin lymphomas, myelosarcoma and multiple myeloma [52]. More recently, ASNase has been used for extranodal NK/T cell lymphoma, nasal type, a highly aggressive neoplasm relatively rare in Europe and North America but more common in Asia and South America [53]. Both ASNases from E. coli and E. carotovora seem effective [54]. A recent phase II study has confirmed that ASNase has an excellent activity on this tumour and, in particular, has suggested that enzymebased treatments are indicated for salvage therapy in patients with disseminated disease, thus far characterised by unfavourable prognosis [55]. Given the presumed relationship between sensitivity to ASNase and low expression of ASNsynt, efforts have been performed to identify other possible targets of antitumour activity. This approach led to the identification of a subset of ovarian cancer cell lines endowed with low ASNsynt mRNA expression and a sensitivity to ASNase greatly enhanced by ASNsynt silencing [56]. More recently, the same group, using a larger panel of ovarian cancer cell lines, has found a stronger correlation between sensitivity to ASNase and expression of ASNsynt at the protein level [57]. In vitro sensitivity to ASNases of subsets of several other solid human neoplasms, such as soft tissue sarcoma [33], hepatocellular carcinoma (Tardito, unpublished results), and gastric carcinoma [58, 59] has also been demonstrated, but the translational relevance of these studies awaits confirmation. In general, access of the tetrameric active enzyme to the tumour microenvironment is expected to be limited, thus preventing a sustained local depletion of Asn and Gln. Hence, modifications of subunit aggregation status to enhance accessibility of the active enzyme to the tumour microenvironment have been recently proposed [48]. PROBLEMS Clinical applications of ASNases are severely restricted by their side effects. First of all, administration of ASNase in childhood ALL may cause coagulative disorders (haemorrhage, disseminated intravascular coagulation (DIC), or thrombotic events), alterations of the gastrointestinal system (loss of appetite, nausea, vomiting), central nervous system symptoms (agitation, depression, hallucinations, disorientation, convulsions, and somnolence or even coma), a mild increase of body temperature, changes in endocrine and exocrine pancreas with acute pancreatitis, and impaired liver function [60]. A number of these drawbacks are probably linked to the glutaminase activity of ASNase [61]. Drug resistance and hypersensitivity, at least in part associated with appearance of neutralising antibodies, can emerge during treatment. Besides causing immune-mediated reactions, which range from mild allergy to anaphylaxis, these antibodies lead to increased clearance of the enzyme by the reticuloendothelial system and to a consequent fast decrease of ASNase activity in plasma [62]. However, correlation between hypersensitivity, duration of remission, and development of specific antibodies in treated patients is still controversial [29, 45, 63, 64]. Therapeutic L-Asparaginases Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 1 SOLUTIONS In general, most studies in the field have aimed at modulating the catalytic activity of ASNase, improving pharmacokinetics, and achieving greater tolerance by structural modifications [65, 66]. Significant enhancements have also been proposed for production, administration, and detection methods, Fig. (2). These data will be reviewed below. Novel Asparaginases It is expected that ASNases with different biochemical characteristics, derived from new or known microorganisms, may be the solution to enzyme toxicity, so that novel, more effective ASNases with less toxic effects are more and more investigated. Recently, a new ASNase was discovered in Helicobacter pylori (H. pylori) by our group [58]. The protein was produced in recombinant form, isolated, purified and characterised. The sensitivity of HL60 leukaemic, cells supports the hypothesis that this enzyme might represent a candidate drug, also considering that its IC50 value is significantly lower than that of E. coli enzyme. The higher cytotoxic effects displayed by H. pylori ASNase compared to the E. coli enzyme are difficult to explain, but this fact underlines the potential of the former as a chemotherapeutic drug for its minimal glutaminase activity, high thermal stability, and maximum activity at physiological pH [67]. Recombinant ASNase derived from W. succinogenes [68] has a very low activity towards Gln and this characteristic 7 may lead to a drastic reduction of side effects. Besides potentially becoming a first line therapy, it could be used to treat patients who have developed hypersensitisation to other microbial ASNases thanks to its low cross-reactivity [14, 69, 70]. Thus, Wolinella ASNase is currently under development through the NIH/NCI-RAID Developmental Therapeutics program, although it has not yet been administered to patients [14]. Particular attention has been given to GLNase-ASNase from Pseudomonas 7A [71], capable of depleting Gln and Asn for prolonged periods. Its considerable antineoplastic activity has been related to the synergic depletion of both amino acids. Pseudomonas 7A GLNase-ASNase appears to be suited for therapeutic use because of its low Km for Gln (micromolar range), good stability and activity in a physiological milieu, and long plasma half-life in tumour-bearing hosts [72, 73]. The widespread problem of neutralising antibodies against non-human protein drugs has led to a great effort in the development of human molecules with therapeutic purposes. Thus, a promising solution to the problem of antiASNase antibodies would be to use recombinant human glycosylasparaginase (also named aspartylglucosaminidase, EC 3.5.1.26) [74]. This enzyme, previously found defective in aspartylglycosaminuria, hydrolyzes the N-glycosidic carbohydrate-to-protein linkage region, aspartylglucosamine, to Laspartic acid and L-amino-N-acetylglucosamine through a reaction mechanism similar to ASNase [75]. Noronkoski and Kelo [76, 77] have demonstrated that the enzyme also has an NOVEL ASNases Pseudomonas A7 H. pylori W. succinogenes Human glycosil-ASNase Clini cal ap pl MODIFICATIONS at ic ASNase for cancer therapy: s ion pH range of activity Endopeptidase resistance PEG conjugation Acylation Bi Innovations DELIVERY ot Reduction of immunoreactivity Erythrocytes Liposomes Human albumin conjugation Increase of activity Rosmarinic acid ec hn ol og y DRUG PRODUCTION Modified hosts ansB- hosts Novel hosts ASSAY Prediction of neutralizing factors Fig. (2). Recent innovations in ASNase usage for cancer therapy: strategies adopted in biotechnology and clinical applications are illustrated. 8 Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 1 Covini et al. ASNase activity similar to the Erwinia enzyme but that, unlike this, glycosylasparaginase lacks glutaminase activity. The characteristics mentioned above make the enzyme a potential anticancer agent. Table 1 summarises the biochemical features of some representative ASNases [40, 58, 69, 72, 76, 78-83], where Km is the substrate concentration at which the reaction rate is half maximal, and kcat or turnover number is the number of catalytic events per second per active site. The former indicates the binding affinity of the enzyme for substrate, the latter represents the maximal rate of conversion obtained at saturating concentration of substrate. In principles, enzymes with a high efficiency have high kcat and low Km and, consequently, a high kcat / Km ratio. Kinetic data can allow some predictions about the performance of ASNase in cancer treatment. Enzymes best suited as drugs would be those with a high activity (high kcat), a high affinity (low Km) for Asn, and a strong preference for Asn over Gln [83]. Among the enzymes used today in the clinics, none is endowed with all these ideal features. Modified ASNases ASNase finds a further application in food industry processes, for example during the preparation of French fries, whose Asn, exposed at high-temperatures, generates acrylamide. For this application, amino acid substitutions of the enzyme sequence have been designed to change the pH range of peak activity [84]. However, the power of this approach is certainly not limited to industrial applications. Structural modifications can be introduced at the amino acid level by site-directed mutagenesis, like in [84], either structure or sequence-based, or by random mutagenesis followed by phenotypic selection [85]. Both can have extraordinary effects on the properties of the wild type protein and are, therefore, a powerful system to modulate both ASNase and Table 1. GLNase activities for therapeutic purposes at will. In this respect, active site transplant by loop grafting is also an alternative, once an appropriate human framework is found. In order to tackle a further mechanism of resistance in clinical sets, a modified E. coli ASNase enzyme has been produced, which is resistant to cleavage by human asparaginyl endopeptidase (legumain), a lysosomal cysteine protease that can cleave native E. coli ASNase [48, 86]. It is expected that the modified enzyme will have a longer halflife in vivo and it will be less allergenic than the native enzyme [87]. Important results have been achieved with pegylated (PEG)-ASNase, a form of E. coli ASNase covalently linked to polyethylene glycol, that decreases immunogenicity of the enzyme and prolongs its half-life [88-90]. Beyond advantages, PEG conjugation has some adverse effects, such as the formation of anti-PEG antibodies, which has led to the development of strategies and methods to select patients who are specifically sensitive to the PEG-conjugated drug and to pursue the selective elimination of anti-PEG antibodies [91, 92]. As an alternative to decrease immunogenicity and avoid acute toxicity, native ASNase has been also acylated, binding palmitoyl residues to epsilon-NH2 groups of lysines [93, 94]. Methods of Administration and/or Delivery Among new administration approaches, an erythrocytecarried ASNase (Graspa®) shows the absence of hypersensitivity reactions. The enzyme can be encapsulated into homologous erythrocytes with two methods: the most widely used requires an incubation under hypotonic conditions or in the presence of membrane-translocating, low-molecularweight protamine; the second method consists in the attachment of the enzyme to the erythrocyte membrane [95]. Biochemical Properties of Representative ASNases. Asparagine Glutamine References Organism kcat (s-1) Km (mM) 0.04-0.07 60 0.04-0.07 60 [78] Erwinia chrysanthemi* 0.058-0.080 397-440 1.7-6.7 65-72 [40, 79] Erwinia carotovora* 0.085-0.098 524-1033 3.0-6.8 2.9-7.6 [80, 81] Arabidopsis thaliana >4 0.23 n.d. n.d. [82] Escherichia coli 0.015 24.0 3.5 0.33 [83] Homo sapiens 0.656 1.09 n.d. n.d. [76] Pseudomonas 7A 0.0046 93.1 0.0044 93.1 [72] Helicobacter pylori 0.290 19.2 46.4 22.1 [58] Wolinella succinogenes 0.0478 166.6 n.d. n.d. [69] Km (mM) Acinetobacter glutaminasificans † † † kcat (s-1 ) In some cases the kcat was calculated from specific activity values reported in the literature. * Taxonomy of the genus Erwinia is in continuous progress, thanks to the introduction of ever new biochemical and genomic methods. Though several Authors report, the two names E. carotovora and E. chrystanthemi as synonyms, this is taxonomically incorrect and the respective ASNases have only a limited amino acid sequence identity (77%, UNIPROT codes: C6DB03 and P06608, respectively). Therapeutic L-Asparaginases Moreover, the improvement in the pharmacokinetics associated with the use of erythrocytes makes it possible to use much lower quantities of enzyme compared to those needed with the free form or with the PEG-conjugated form, thus reducing toxicity risks [96]. Palmitoyl-conjugated ASNase can also be encapsulated in large liposomes (simplified Dehydration-Rehydration Vesicles, sDRV; median diameter 1.249 nm) [97, 98] and in small liposomes (extruded vesicles, VET: median diameter 158-180nm). Both devices mitigate anaphylactic reactions and enhance antitumour activity, with VET also prolonging the circulation time of the enclosed enzyme [97, 98]. Poznansky and his group have described the advantage of ASNase cross-linked to homologous albumin in a polymeric form. This modification leads to an enzyme much more resistant to proteolytic degradation and less immunogenic than the free enzyme, due to the masking of antigenic determinants by albumin. Moreover, immobilisation of ASNase with homologous albumin reduces the dose needed for antileukaemia activity [99, 100]. The co-administration of the natural polyphenol rosmarinic acid with E. coli ASNase can significantly improve, at least in vitro, the activity of ASNase when the concentration of rosmarinic acid is between 0.375 x 10-4 and 3.0 x 10-4g/L [101]. Drug Production A group of recent patents concerns biotechnological techniques for production of recombinant ASNases, with particular attention to the improvement of methods and expression vectors. The presence of the gene encoding an ASNase II subunit, native to the chromosome of E. coli host strains, could have been a potential obstacle and several methods have been implemented to circumvent this problem. In one, the host cell chromosome has been modified to encode the same ASNase II cloned in the plasmid for recombinant protein expression [102]; in another, the E. coli genome has been modified to delete native ASNase genes [103, 104]. An alternative method to produce E. coli ASNase exploits the construction of the encoding pNAN5 plasmid and its transformation in Bacillus cereus 1676 cells. This process allows improvement of industrial production and purification of the enzyme [105]. Assays Methods to monitor activity of serum ASNase to tailor the therapeutic dose to the single patient are currently used in research settings, but have not been validated for patient care. Inactivation of ASNase by neutralising factors present in patients' serum is not necessarily accompanied by clinical signs and, therefore, can remain undetected. For this reason, a quick and easy-to-use test for predicting the presence of ASNase neutralising factors, mainly antibodies, in patient serum has been proposed so as to adjust the dose of administered enzyme or to replace the ASNase used with another enzyme not sensitive, or less sensitive, to the neutralising factors [106]. Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 1 9 CURRENT & FUTURE DEVELOPMENTS Thanks to more than one century of studies, it is now clear that Asn and Gln are pivotal in normal metabolism. On the other hand, recent work on amino acid metabolism in cancer has made ASNase and related enzymes potentially key therapeutic tools in previously untested tumours. Moreover, the search for natural variants and the production of novel modified forms of ASNase by protein engineering appear as the most powerful approaches to improve the therapeutic potential of the enzyme. Recent studies on human L-ASNases [74, 77, 107, 108] would suggest that these molecules will be the most likely to be exploited in future clinical applications. However, their distribution, limited to immunologically privileged tissues (brain and testis), along with the discovery that ASNase is an autoantigen in rats [108] indicate that this route should be undertaken with caution. Alternatively, novel sources of ASNases, such as plants, have not yet been fully considered. In this respect, a bioinformatic analysis for immunogenicity of representative ASNases from different species was performed by Ellipro [109] and CBTOPE [110], Table 2. Ellipro [109] predicts linear and discontinuous antibody epitopes based on a protein antigen's 3D structure or a model generated from a protein sequence [111, 112]. CBTOPE [110] discriminates the antibody epitope residues and non-epitope residues for a given protein sequence by using the amino acid composition generated from the query sequence(s). Our result suggests that the number of predicted epitopes for several plant enzymes is similar to that of bacterial enzymes, Table 2. Moreover, immunodominant epitopes can be tackled by structural modifications [40]. As an example, in Fig. (3a,b,c) epitopes predicted by Ellipro [109] are mapped in dark grey on the molecular surface of E. coli ASNase (Protein Database ID: 3ECA). Linear epitopes demonstrated to be biologically relevant according to [111] (§) and [112] (*) are shown in black. Biologically relevant epitope 115-124 was also predicted by Ellipro. In panel D, discontinuous epitopes predicted by Ellipro [109] are represented. New modifications are also under development. For example, Zhang and his group conjugated E. coli ASNase to silk fibroin and silk sericin from Bombyx mori, using a crosslinker [113]. The modified enzyme has enhanced thermal and storage stability, resistance to trypsin digestion, a lengthened circulatory half-life in vitro, and reduced immunogenicity. Only recently, E. chrysanthemi L-asparaginase has been pegylated [114], while the capacity of ASNase to deplete asparagine has been combined with the inhibitory activity of a small interfering RNA (siRNA) towards ASNsynt [115]. It is likely that the combination of different approaches mentioned above will sort out the issues related to the clinical usage of ASNase reasonably soon. ACKNOWLEDGEMENTS Financial support for all the authors derive from the Italian Ministry for University and Research. ST is a Rina Fallini scholar of the Medical Faculty of the University of 10 Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 1 Table 2. Covini et al. Number of Epitopes Predicted for L-Asparaginase from Different Species. ElliPro [109] Uniprot code CBTOPE [110] Epitope Prediction Organism Epitope Prediction Linear Discontinous P20933 Homo sapiens 3 4 19 Q7L266 Homo sapiens 2 2 22 P06608 Erwinia chrysanthemi 5 4 29 CSDB03 Erwinia carotovora 13 8 23 P00805 Escherichia coli K12 4 5 21 B6ZCD8 Helicobacter pylori CCUG 4 14 24 O34245 Wolinella succinogenes 17 3 Q8GXG1 Arabidopsis thaliana 3 12 19 Q9ZSD6 Lupinus luteus 4 chain A 12 chain A 15 4 chain B 14 chain B 4 14 (Genbank) CAE11777.1 Cavia porcellus A 44 B 304-313 [*] 244-265 115-124 [*] 14-52 75-81 55-58 [§] 311-318 C D 260-261 264-265 198-211 1-2 [§] 19-20-120 137-144 181-195 15-18 14-30-31 Fig. (3). Epitopes mapped on the atomic surface of E. coli ASNase (Protein Database ID: 3ECA). Panels A, B and C: linear epitopes mapped on subunit B (white) of the tetramer. Dark grey islands: epitopes predicted by Ellipro [109]. Black islands: linear epitopes demonstrated to be biologically relevant according to [111] (§) and [112] (*). 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